ANDROGENS INFLUENCE SEXUAL-DIFFERENTIATION OF EMBRYONIC MOUSE HYPOTHALAMIC AROMATASE NEURONS IN-VITRO

Estrogen formed perinatally in the brain from testicular androgen by a romatase is involved in the irreversible determination of male brain d evelopment. Perinatal sex differences in aromatase activity have been observed in the hypothalamus. Testosterone (T) is a major modulator fo r aromatase in the adult rat hypothalamus. However, it is not known wh ether circulating T influences aromatase neurons during fetal brain de velopment. To study the influence of androgen exposure on embryonic ne uronal aromatase, gender-specific primary cell cultures were prepared from embryonic day 15 mouse hypothalamus and cortex. Estrogen formatio n by cultured neurons was measured using an in vitro (H2O)-H-3 product formation assay, and aromatase neurons were identified by immunocytoc hemistry using a highly specific antiserum. Aromatase activity (AA) pe r well and numbers of aromatase-immunoreactive (IR) neurons per microt ubulus associated protein II-IR neurons x 10(5) were significantly hig her in male hypothalamic cultures compared with female when grown in t he absence of sex steroids. When AA was calculated per aromatase-IR ne uron, no differences in enzyme activity were found between male and fe male. Therefore, the level of AA in individual male hypothalamic neuro ns is similar to the female, but a higher proportion of male neurons e xpress aromatase. After T treatment, AA per well (P less than or equal to 0.001) and AA/aromatase-IR cell (P less than or equal to 0.005) in male and female hypothalamic cultures was significantly increased us. controls. In addition, numbers of aromatase-IR neurons/microtubulus a ssociated protein II-LR neurons x 10(5) were significantly higher afte r T exposure compared with controls (P less than or equal to 0.001). A ndrogenic effects on hypothalamic AA and aromatase-IR cell numbers wer e dose-dependent and mediated via androgen receptor stimulation, since the observed effects were inhibited by the androgen-receptor antagoni st flutamide. There was no effect of T on cortical AA or aromatase-IR cell numbers, indicating area-specific regulation of brain aromatase. We conclude that 1) sex differences in hypothalamic AA are due to a hi gher percentage of neurons expressing aromatase in males rather than t o higher AA in individual male hypothalamic aromatase-IR cells, and 2) androgens influence the development of the fetal hypothalamic aromata se system. Because T influenced both the embryonic male and female hyp othalamic neurons in culture, the developing mouse brain aromatase app ears to be bipotential in response to androgen. The data suggest that environmental and genetic factors affecting androgen level and/or andr ogen receptor function in the developing brain could interfere with th e sexual differentiation of estrogen forming neurons.